1. The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice
by Peter J. Turnbaugh, Vanessa K. Ridaura, Jeremiah J. Faith, Federico E. Rey, Rob Knight, and Jeffrey I. Gordon
Sci Transl Med
Volume 1(6):6ra14-6ra14
November 11, 2009
Copyright © 2009, American Association for the Advancement of Science

2. Fig. 1 Design of human microbiota transplant experiments.
Design of human microbiota transplant experiments. (A) The initial (first-generation) humanization procedure, including the diet shift. Dark red arrows indicate fecal collection time points. (B) Reciprocal microbiota transplantations. Microbiota from first-generation humanized mice fed LF/PP or Western diets was transferred to LF/PP diet– or Western diet–fed germ-free recipients. (C) Colonization of germ-free mice starting with a frozen human fecal sample. (D) Characterization of the postnatal assembly and daily variation of the humanized mouse gut microbiota. (E) Sampling of the humanized mouse gut microbiota along the length of the gastrointestinal tract.
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science

3. Fig. 2 The effects of switching from the LF/PP diet to the Western diet on the humanized mouse gut microbiota.
The effects of switching from the LF/PP diet to the Western diet on the humanized mouse gut microbiota. (A) 16S rRNA gene surveys (analyzed by unweighted UniFrac-based PCoA) from the human donor (green), first-generation humanized mice fed LF/PP (red) or Western (blue) diets, second-generation microbiota transplant recipients consuming the LF/PP (teal) or Western (purple) diets, and mice humanized with a frozen sample fed LF/PP (yellow) or Western (orange) diets (total of 340 samples with >800 sequences per sample). Weighted UniFrac resulted in a similar overall clustering pattern (data not shown). Principal coordinate 1 (PC1) and PC2 are the x axis and y axis, respectively, and have been scaled on the basis of percent variance. PC3 is depicted by the shading of each point. The percent variance explained by each coordinate is shown in parentheses. dpc, days after colonization with a human donor sample; dpd, days after diet switch. (B) Taxonomic distribution [Ribosomal Database Project (RDP) level 3 (class-level taxa) (27)] of two generations of humanized mice fed a LF/PP or Western diet. Values represent the average relative abundance across all samples within the indicated group. c, cecal samples (whereas all other samples are fecal); M, month. (C) 16S rRNA gene sequences (analyzed by unweighted UniFrac-based PCoA) from the mice in the experiment described in Fig. 1A. The x and y axes are scaled on the basis of the percent variance accounted for by each component (shown in parentheses). Each box corresponds to a single time point.
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science

4. Fig. 3 Postnatal assembly of the humanized gut microbiota.
Postnatal assembly of the humanized gut microbiota. (A) Rarefaction curves measuring bacterial diversity in the fecal communities (species-level phylotypes defined by ≥97% identity). The curves are based on V2 16S rRNA gene sequences obtained from mice before weaning (P14) and after weaning (P28). Values are mean ± 95% confidence interval. (B) Taxonomic distribution [RDP level 3 (27)] of the gut microbiota sampled from mice from P14 to P85. Values represent the average relative abundance across all samples within a given group.
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science

5. Fig. 4 Clustering and taxonomic analysis of the gut microbiota of humanized mice consuming a LF/PP or Western diet.
Clustering and taxonomic analysis of the gut microbiota of humanized mice consuming a LF/PP or Western diet. (A) 16S rRNA gene surveys (analyzed by unweighted UniFrac PCoA) of the humanized microbiota along the length of the gut (n = 148 samples with >500 sequences per sample). Weighted UniFrac results in a similar overall clustering pattern. PC1 and PC2 are the x axis and y axis, respectively. The percent variance explained by each coordinate is shown in parentheses. (B) Taxonomic distribution [RDP level 3 (27)] in communities distributed along the length of the gut. Values represent the average relative abundance across all samples within a given group. SI, small intestinal segment.
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science

6. Fig. 5 Clustering of the distal gut microbiome, the C
Fig. 5 Clustering of the distal gut microbiome, the C. innocuum SB23 transcriptome, and the community meta transcriptome in the ceca of humanized mice.
Clustering of the distal gut microbiome, the C. innocuum SB23 transcriptome, and the community meta transcriptome in the ceca of humanized mice. Microbiome or transcriptome profiles were normalized by z score, used to construct a correlation distance matrix, clustered with UPGMA (unweighted pair group method with arithmetic mean), and visualized (see Supplementary Material; Matlab version 7.7.0). (A) Clustering of fecal microbial gene content in the human donor’s microbiome and two groups of humanized mice starting at day 1 after colonization (n = 3 to 5 mice per group; fecal DNA was pooled before sequencing). All mice were maintained on the LF/PP diet (red) for 28 days, at which point group 2 was transferred to the Western diet (blue). (B) Clustering of C. innocuum SB23 gene expression in humanized mice fed the LF/PP (red) or Western (blue) diet. (C) Clustering of the gut microbiome’s meta transcriptome in humanized mice fed a LF/PP (red) or a Western (blue) diet. Black circles represent validated clusters (inconsistency threshold = 0.75, “cluster” function in Matlab version 7.7.0). (D) qRT-PCR validation of C. innocuum SB23 gene expression in humanized mice (n = 3 to 5 samples per group; see Supplementary Material). Mean values ± SEM are plotted (*P < 0.05, Student’s t test).
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science

7. Fig. 6 Transmissibility of adiposity from humanized mice to germ-free recipients.
Transmissibility of adiposity from humanized mice to germ-free recipients. (A) The effects of Western and LF/PP diets on epididymal fat pad weight (expressed as a percentage of total body weight) in humanized gnotobiotic mice (n = 5 to 8 mice per group; n = 2 independent groups). (B) Percent increase in total body fat (measured by DEXA) after colonization of germ-free mice with a cecal microbiota harvested from humanized donors fed the Western or the LF/PP diet (n = 4 to 5 mice per group). Recipients were fed a LF/PP diet. Mean values ± SEM are plotted (*P < 0.05, Student’s t test).
Peter J. Turnbaugh et al., Sci Transl Med 2009;1:6ra14
Copyright © 2009, American Association for the Advancement of Science